HLA-DO / H2-O (DO) is a non-classical MHCII protein that functions as a specific competitive inhibitor of the peptide exchange factor HLA-DM / H2-M (DM), regulating peptide loading onto MHCII molecules in antigen- presenting cells. During CD4 T cell development, thymocytes are selected for conversion to conventional nave CD4 T cells or self-tolerant regulatory T cells, or are deleted by negative selection, depending on the strength of interaction with MHCII-bound peptides displayed on antigen presenting cells in the thymus. We eluted peptides from wild-type C57BL/6 and DO-knockout thymus, and found alterations in the spectrum of peptides presented by MHCII molecules. We examined CD4 T cell development in DO-deficient mice, and found alterations in Treg number and function, and increased sensitivity to viral infection. The overarching hypothesis of this proposal is that DO regulates antigen presentation by restraining the peptide editing activity of DM in order to allow a broad representation of peptides presented at the cell surface, and this activity is essential for proper thymic selection of self-tolerant conventional and regulatory T cells. There are two specific aims. Aim 1 is to evaluate the role of DO-dependent antigen presentation in selection of CD4 Tconv and Treg populations. We will identify peptides differentially presented in the thymus in the presence or absence of DO, follow individual TCR clonotypes as they undergo thymic selection in WT and DO-KO TCR-restricted mice, combine these results to identity DO-peptides responsible for Treg skewing, and finally characterize functional effects of Treg dysregulation in DO-KO mice. These experiments will test the hypothesis that narrowing of the peptide repertoire presented by MHCII molecules induced by deletion of DO results in altered thymic selection and peripheral regulation of regulatory T cells. Aim 2 is to determine the role of HLA-DO / H2-O in regulating Tconv and Treg populations in an infection model. We will evaluate the functional consequences of DO-induced changes in the TCR repertoire and activation state of CD4 T cell populations in the response of BL/6 mice to primary influenza infection. Tregs have been shown previously to regulate CD4 and CD8 T cell responses and recruitment of innate effector cells, and we observe alterations in these effects in DO-KO mice, suggesting a dysregulation of Tregs in the absence of DO. We will test this hypothesis, using a FoxP3-DTR system to swap Treg compartments between mice. A second part of this aim is to evaluate the functional consequences of DO- induced alterations in antigen presentation and TCR repertoires using the influenza infection model. The long- term goals of this project are to define how control of DM-mediated peptide editing by DO regulates selection of self- and pathogen-derived peptides displayed on MHCII, and to decipher the impact of DO-regulated peptide presentation on the development and function of the CD4 T cell repertoire. 1